This project will test two overarching hypotheses about bioenergetic work in respiratory bacteria, i.e., membrane-associated processes that are energized by an electrochemical gradient of protons across the membrane, the protonmotive force (pmf). The experimental system for much of the work is an alkaliphilic Bacillus that exhibits constitutive readiness to meet the challenge of a high bioenergetic work-load. Alkaliphilic Bacillus pseudofirmus OF4 also has special adaptations that facilitate its bioenergetic work under conditions of low pmf. The first overarching hypothesis is that specific structural features enhance the ability of the alkaliphile ATP synthase and its uniquely complex Mrp-type Na? antiporter to respectively support respiration- dependent ATP synthesis and cytoplasmic pH homeostasis at low pmf. Adaptive features are hypothesized to enable these pmf-user complexes to take advantage of sequestered paths of proton transfer from respiratory pumps and to gather protons in proton-poor environments. Other features of bioenergetic machinery, including ATP synthase, prevent proton leaks during oxidative phosphorylation (OXPHOS). The first two Specific Aims focus on structural-functional features of alkaliphile machinery. Specific Aim #1 tests the hypothesis that the unique P51XXEXXP motif of the F1F0-ATP synthase c-rotor prevents proton leakiness. This will be assessed through comparative structural studies of the stable c-rotor rings from wild-type and a cP51A mutant. 2-D projection maps will be analyzed and high resolution structural information from 3D crystals will be sought. Specific Aim #2 tests the hypothesis that the 7-protein Mrp hetero-oligomer is a consortium of antiporters and other transporters that are interdependent and synergistic, e.g. jointly presenting a large external surface engineered for proton-gathering. Proposed experiments include a screen for a putative anion transport function using a new Mrp mutant that confers high Na+resistance but lacks antiport activity. The second overarching hypothesis is that major pmf-consuming complexes such as ATP synthase and Mrp antiporter draw upon a network of cell components and regulators to meet bioenergetic challenges. In non-alkaliphiles that are not hard-wired for heavy bioenergetic work-loads, activation of a major pmf-consumer by sodium-alkali challenge elicits a systems response that facilitates management of challenge. The third and fourth Specific Aims focus on physiological contexts of bioenergetic work. Specific Aim #3 probes the interplay between alkaliphile respiratory chain supercomplexes, cardiolipin and ATP synthase in OXPHOS at low pmf. Specific Aim #4 will model and test a hypothesized systems response to Mnh (a Mrp homologue) activity in Staphylococcus aureus that results in a net increase in the transmembrane potential and impacts membrane lipid fluidity as well as sensitivity to antimicrobial peptides. The model will be tested and built via a panel of genetic, biochemical and molecular assays. The overall goal is to fill in gaps in our understanding of the mechanisms and plasticity of bioenergetic work and of the cell-wide responses that manage changes in bioenergetic work-load.